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Gut, 1974, 15, 260-267
Collagen proline hydroxylase activity and 35S
sulphate uptake in human liver biopsies
J. O'D. McGEE, R. S. PATRICK, MARION C. RODGER, AND
CAROLYN M. LUTY
From the University Department of Pathology, Royal Infirmary, Glasgow
SUMMARY In an effort to assess connective tissue biosynthetic activity in human liver disease,
collagen proline hydroxylase (a key enzyme in collagen biosynthesis) and the uptake of 35S sulphate
(a precursor of sulphated mucopolysaccharides) were measured in hepatic tissue obtained mainly by
percutaneous biopsy.
A procedure is described for the quantitation of collagen proline hydroxylase in cryostat sections
which allows for the simultaneous histopathological examination of the liver specimen. A three to
eightfold increase in the activity of this enzyme was found in four cirrhotic livers compared with the
mean value of four normal livers and two biopsies from patients with Gilbert's syndrome. Elevated
hydroxylase levels were found also in five patients with hepatic dysfunction but without cirrhosis
(four alcoholics and one patient with persistent hepatitis associated with serum smooth muscle
antibody). It is suggested that the hepatic level of collagen proline hydroxylase may be a useful
quantitative index of fibroblastic activity in human liver disease.
Autoradiographic studies of radioactive sulphate uptake in biopsy specimens from patients with
chronic liver disease showed an exaggerated incorporation of isotope not only at sites of established
fibrogenesis but also in the walls of sinusoids throughout the liver.
Active hepatic fibrogenesis compared with passive
stromal condensation may be involved in the progression of some liver diseases to cirrhosis. This
implies an increase in the biosynthesis within the
liver of the connective tissue components, collagen
and mucopolysaccharide (Galambos, 1966; Popper
and Udenfriend, 1970). Morphological examination
of liver can determine the presence or absence of
hepatic fibrosis, but it is impossible by this means to
assess the amount of collagen or mucopolysaccharide being produced at any given point in time.
Similarly, it is not feasible to use the standard isotopic procedures for measuring collagen and mucopolysaccharide synthesis in percutaneous needle
biopsies of liver because of the limited amount of
tissue available.
It has been reported that the activity of collagen
proline hydroxylasel, the enzyme responsible for
hydroxyproline biosynthesis in collagen, generally
'Collagen proline hydroxylase has also been referred to as prolylhydroxylase, protocollagen proline hydroxylase, and peptidyl proline
hydroxylase.
Received for publication 19 December 1973.
reflects the rate of collagen synthesis in a variety of
tissues (see Grant and Prockop, 1972, for review).
It has also been shown that the uptake of 35S sulphate determined autoradiographically reflects the
rate of sulphated mucopolysaccharide synthesis in
experimental liver disease (McGee and Patrick,
1969a and b). Since it seemed possible that this
enzyme and 35S sulphate uptake could be measured
in needle biopsies of liver taken for diagnostic purposes, the present investigation was undertaken with
a small number of normal specimens and with other
specimens from a variety of hepatic diseases. The
techniques used allow for simultaneous histopathological examination of the biopsy material.
Materials and Methods
3,4 3H-proline (specific activity 38Ci per mole) was
purchased from New England Nuclear Enterprises,
Frankfurt, Germany; 35S sulphate (specific activity
100 mCi per mole) from the Radiochemical Centre,
Amersham, England; minimum essential medium
from Biocult, Paisley, Scotland; Triton X-100 and
260
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Collagen proline hydroxylase activity and 35S sulphate uptake in human liver biopsies
ferrous ammonium sulphate from British Drug
Houses, Liverpool, England; all other reagents
from Sigma, London, England.
Most of the liver biopsies were carried out by the
percutaneous route using a Menghini needle and
a few by open surgical operation.
MEASUREMENT OF COLLAGEN PROLINE
HYDROXYLASE ACTIVITY
Entire needle biopsy specimens were frozen immediately onto a metal cutting block on top of a
layer of partially frozen 0.15 M NaCl by immersing
the base of the chuck in a mixture of ethanol and
solid CO2. Surgical wedge biopsy specimens were
trimmed to exclude the edges of excision and the
liver capsule before freezing in the same manner. A
total of seven or 12 sections were cut from the
needle and surgical specimens respectively in a cryostat maintained at - 15°C. The first and last sections
(8 ,um thick) were processed for histological examination to determine the purity of the samples used
for assay; those contaminated with other tissues,
such as skeletal muscle or large pieces of blood clot,
were excluded from the study. The intervening unthawed sections (20 ,tm thick) were used for the
measurement of collagen proline hydroxylase activity. These were transferred by a cold needle to a
1 ml glass-teflon homogenizer which was stored in
the cryostat on ice. The sections were homogenized
at 4°C in 0.5 ml of 0.05 M Tris-HCI, pH 7.2, containing 025 M sucrose, 10-5 M ethylenediaminetetraacetic acid, 10-3 M dithiothreitol, 0.1 % Triton
X-100, and 50 gg/ml of phenylmethylsulphone
fluoride. Aliquots of the homogenate were assayed
in duplicate for enzyme activity by the tritium release
method using a 3,43H-proline-labelled substrate
(Hutton, Tappel, and Udenfriend, 1966); these
assays were performed immediately since the activity of the enzyme rapidly deteriorates in crude liver
homogenates. The reaction mixture for the assay
contained the following reagents in a final volume of
1 ml: 50 ,gmoles of Tris - HCl (pH 7.2); 5 ,umoles of
ascorbate; 1 ,umole of ferrous ammonium sulphate;
2 mg of heat-denatured bovine serum albumin;
0.4 mg of catalase; 1 ,umole of x-ketoglutarate; 0-1
,umole of dithiothreitol; 0.1 ml of 3H-proline-labelled
substrate; 0 05-0.20 ml of liver homogenate as
enzyme. The complete mixture was incubated at
30°C and the reaction terminated after 30 minutes
by the addition of 0.1 ml of 50 % trichloroacetic acid.
The concentration of Fe++, ascorbate, and a-ketoglutarate is 10 times higher than that usually employed (McGee, Langness, and Udenfriend, 1971)
in order to increase the sensitivity of the assay. It has
been shown that the tritium released into water in
this assay is proportional to the formation of hy-
261
droxyproline (Hutton et al, 1966). Separate aliquots
of the homogenate were taken for protein estimation
by the method of Lowry, Rosebrough, Farr, and
Randall (1951) using bovine serum albumin as a
standard. The collagen proline hydroxylase activity
of each biopsy specimen was expressed as counts per
minute (cpm) released from the tritium-labelled substrate per milligram of tissue protein in the homogenate. Tissue protein was chosen as the reference
point for enzyme activity rather than tissue mass
since it is impracticable on a routine basis to weigh
cryostat sections. Variation in protein concentration
of the homogenates did not affect the linearity of the
enzyme assay (see results).
The tissue remaining after sections had been taken
for the hydroxylase assay was processed routinely
for histopathological examination. It was entirely
adequate in size and preservation for diagnostic
purposes.
35S SULPHATE UPTAKE
Immediately after the biopsy specimen was taken a
small piece (3 mm x 1 mm) was cut from each end
and placed in 5 ml of HEPES-buffered minimum
essential medium, pH 7.2, containing 10-3M ascorbate, 10-6M ferric nitrate, and 250 ,uCi of 35S sulphate in a sterile universal container. The tissue was
incubated at 37°C for 90 minutes in a 95 % 02, 5 %
C02 atmosphere. After incubation the medium was
decanted, the tissue washed three times with 10%
buffered formalin, pH 7.0, and fixed in the same
solution for 16 hours. Thereafter it was processed
routinely to paraffin and autoradiographs were prepared using Ilford G5 nuclear track emulsion. The
autoradiographs were exposed for six weeks, developed by routine methods, and then stained with
neutral red.
Results
The procedure used for the measurement of collagen
proline hydroxylase activity in human liver was
worked out initially on a large sample of tissue
obtained from a renal transplant donor immediately
after death. After excision the liver was immersed in
liquid nitrogen and stored at - 80°C. Of the various
homogenization conditions used for the measurement of hepatic collagen proline hydroxylase it was
found that 0-05 M Tris-HCl (pH 7-2) containing
0-1 % Triton and 10-3 dithiothreitol yielded optimum
enzyme activity (table I). This buffer was used
throughout the study. As shown in fig 1 the release
of tritium from 3,4 3H-proline-labelled substrate is
directly proportional to the amount of liver homogenate. The linearity of the reaction was not affected
by changes in the protein concentration of the
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J. O'D. McGee, R. S. Patrick, Marion C. Rodger, and Carolyn M. Luty
262
Collagen Proline
Additions to Buffer'
Hydroxylase
(cpm/mg protein)
10-'M Dithiothreitol
10-'M Dithiothreitol + 0-1 % Triton X-100
10-'M Dithiothreitol
10-'M Dithiothreitol + 01 % Triton X-100
266
883
297
1126
Table I Hepatic collagen proline hydroxylase activity
in different homogenization buffers
'In each experiment the buffer consisted of 0.05 M Tris-HCI (pH 7.2)
containing 0.25 M sucrose, 10-'M EDTA, and phenylmethylsulphonic
fluoride to a concentration of 50 ,tg/ml. Additions to this buffer were
made as indicated. An approximately equal weight of human liver was
homogenized in 40 volumes of each buffer and aliquots were assayed
immediately for collagen proline hydroxylase activity.
results of assaying an aliquot of this enzyme preparation with the same substrate on five consecutive days were 951, 1081, 1120, 980, and 901 cpm.
Collagen proline hydroxylase activity in four normal
livers, in two cases of Gilbert's syndrome, and in four
examples of hepatic cirrhosis are recorded in table II.
Diagnosis'
Protine Hydroxylase (cpm/mg protein)*
Normal
Gilbert's disease
1158 (726, 893, 1459, 1553)
1310 (946, 1674)
Cirrhosis'
Mildly active
Moderately active
Aggressive
3585
6000
9366 (9144, 9588)
Table II Collagen proline hydroxylase activity in
homogenates over the range 0-5-2.2 mg/ml. Tri- histochemically normal liver and in cirrhosis
tium release is entirely dependent on a-ketoglutarate 'The diagnosis in each instance was established by clinical and histo(fig 1), a cofactor not required by other hepatic pathological criteria.
mean value is shown for each group where more than one patient
hydroxylases. The variability of the assay in this 'The
was studied. The individual values within each group are shown in
laboratory was determined by measuring the acti- brackets.
vity of partially purified mouse skin collagen proline 'See figs 2a-c.
hydroxylase (Rhoads and Udenfriend, 1970). The
The normal liver values were derived from patients
with no biochemical or histological hepatic abnormality; two were undergoing operation for staging
of Hodgkin's disease (726 and 1459 cpm/mg), the
third was a psychotic patient with minimal hepatomegaly (893 cpm/mg), and the fourth was the transplant donor referred to above (1553 cpm/mg). The
CL
mean value for those patients with Gilbert's synUdrome (serum bilirubin 3 mg/100 ml)- a hepatic
a 400disorder not associated with connective tissue formation-fell within the normal range. In contrast,
the proline hydroxylase activity in four patients
with cirrhosis was several-fold higher than normal.
The case of cirrhosis, which was aggressive (fig 2a) by
standard histological criteria (Popper, 1966), had an
200
enzyme value six times higher than the highest control, while that of moderately active cirrhosis (fig 2b)
E
and of mildly active cirrhosis (fig 2c) were four- and
two-fold higher than the highest control.
Of particular interest was a small group of patients with raised hepatic collagen proline hydroxylase activity who did not have cirrhosis, and some
of whom had little or no morphological evidence of
100
200
collagen deposition at the time of biopsy. The enzyme
HOMOGENATE (Pj)
levels together with the morphological diagnosis and
Fig 1 Dependence of collagen proline hydroxylase
the degree of fibrosis in these cases is shown in
assay on homogenate concentration and oc-ketoglutarate.
table III. Patients 1-4 had in common a history of
The protein concentration of the liver homogenate was
chronic alcoholism and all had at least one frankly
1.9 ,ug/ml. Hydroxylase activity was measured as tritiated abnormal liver function test on admission; all had
water formed by incubation of the aliquot indicated with
proline hydroxylase values several times normal. The
3,4 3H-proline labelled substrate.
main clinical finding in patients 1 and 2 was hepatoTritiated water formed in the presence of oc-ketoglumegaly. Patients 3 and 4 had marked portal and
tarate *-*
periportal fibrosis together with the other morphoTritiated water formed with 200 ,ul of liver homogenate
logical and clinical stigmata of alcoholic liver disease.
in the absence of a-ketoglutarate E
a
0
0
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Collagen proline hydroxylase activity and 35S sulphate uptake in human liver biopsies
263
4
Fig 2b
Fig 2a
Fig 2 Light micrographs of (a) aggressive, (b) moderately active, and (c) mildly active cirrhosis. In (a) there
is marked chronic inflammatory cell infiltration offibrous septa, piecemeal necrosis atd fine (immature) collagen
fibre deposition (arrows) at the periphery of regenerative nodules. In (b) there is inflammatory cell infiltration of the
septa with slight erosion of the edge of regenerative nodules. The collagen proline hydroxylase activity found in these
biopsies expressed as cpm/mg protein in the liver homogenate was (a) 9144, (b) 6000, and (c) 3585. (a) Haematoxylin
and Van Gieson's stain, x 225; (b and c) haematoxylin and eosin, x 140.
Fig 2c In (c)
the fibrous septa
consist mainly
of thick (mature)
collagen bundles
and there is little
inflammatory
cell infiltrate.
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J. O'D. McGee, R. S. Patrick, Marion C. Rodger, and Carolyn M. Luty
264
phated mucopolysaccharide biosynthesis was the
uptake of 35S sulphate. Representative results from
(cpm/nmg protein) a study of nine cases of chronic liver disease are
illustrated in figures 3-5. A case of congenital he2966
1
Nil
Fatty change
3027
Nil
2
Intrahepatic cholestasis
patic fibrosis, an example of non-progressive hepatic
3
2981
Alcoholic hepatitis
++
fibrosis, was used as a control in this series. As shown
4
4255
++
Alcoholic hepatitis
5
5166
Nil
Persistent hepatitis
in fig 3b there is little uptake of 35S04 except in
a few cells in the connective tissue bands and in
Table III Collagen proline hydroxylase activity in
isolated sinusoidal cells. This is comparable to the
non-cirrhotic liver disease
degree of cell labelling in normal liver. In compari'Fibrosis was assessed morphologically. Patients 3 and 4 had moderate
son, the uptake in vitro of the isotope in aggressive
portal and periportal fibrosis.
liver disease typified by primary biliary cirrhosis and
acute alcoholic hepatitis is much more marked (figs
Patient 5 had a persistent hepatitis with intra- 4b and 5). It is pronounced at the sites of connective
hepatic cholestasis, but no evidence of hepatic tissue formation and in the portal tracts. Rather
fibrosis at the time of biopsy. She also had a serum surprisingly, however, the isotope is also avidly
smooth muscle antibody but no antimitochondrial taken up by numerous sinusoidal cells throughout
antibody. This woman, possibly a developing case the liver. Mast cells, which are known to incorporate
of active chronic hepatitis, had a hydroxylase level 35S sulphate, were inconspicuous in these biopsies as
five times higher than the mean normal value.
determined by toluidine blue staining, and could not
The method adopted for the assessment of sul- account for the results obtained.
Patient
Diagnosis
Fibrosis'
Collagen Proline
Hydroxylase
Fig 3b
Fig 3a
Fig 3 Liver in congenital hepatic fibrosis. (a) The light micrograph shows numerous small bile ducts in mature fibrous
septa. H and VG, x 159. (b) The autoradiograph shows uptake of 35S sulphate confined to a few mesenchymal cells
(arrows) in the fibrous bands and to mucinous material in bile ducts, x 159.
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265
Collagen proline hydroxylase activity and 35S sulphate uptake in human liver biopsies
..
;4t
'*
C
C
S.,~~~~~~~~~~~~~~W
....,i,,X,.E~
~ ~~~i,
ig4 5% Lie in alohli
hptitis
Fig 4a
Fig 4b
Fig 4 Liver in primary biliary cirrhosis. (a) The light micrograph shows chronic portal and periportal inflamimation wtith
fibrosis and an abnormal dilated interlobular bile duct. H and E, x 105. (b) The autoradiograph shows considerable
grap of
ilutaesantbe
ereo
uptake of 35S sulphate at the sites of connective tissue deposition. There i salsoA.&
labelling
many hepatic sinusoidal
cells, x 105.
Fig 5 Liver in alcoholic hepatitis
*with fatty change. This autoradiograph illustrates a notable degree of
X
35S sulphate uptake by sinusoidal cells
(arrows) in this form of chronic
aggressive liver disease, x 560.
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266
J. O'D. McGee, R. S. Patrick, Marion C. Rodger, and Carolyn M. Luty
Discussion
Collagen proline hydroxylase, an enzyme specific to
the collagen pathway, catalyses the biosynthesis of
hydroxyproline, an amino acid found only in collagen and to a very limited extent in elastin, by
hydroxylating certain prolyl residues in growing
collagen oc chains (Miller and Udenfriend, 1970). It
does not hydroxylate free proline (Hutton and
Udenfriend, 1966) but only peptidyl proline occurring in sequences of the general structure -X-ProGly- where X can be one of several amino acids
(McGee, Rhoads, and Udenfriend, 1971); the bulk
of the collagen molecule is composed of repeating
-X-Pro-Gly- triplets. Collagen proline hydroxylase
requires as cofactors for its action 02, Fe++, ox-ketoglutarate, and a reducing reagent such as ascorbate.
It is the prototype of a new class of oxygenases
which stoichiometrically decarboxylate ox-ketoglutarate during the hydroxylation process (Udenfriend,
1970). In this respect it differs from the many other
hepatic hydroxylases. The fact that the enzyme
activity assessed in this study was totally dependent
in the presence of a-ketoglutarate indicates that only
collagen proline hydroxylase activity was measured.
Several observations suggest that the tissue level
of collagen proline hydroxylase is in general an indicator of fibroblastic biosynthetic activity. In physiological conditions where collagen production is elevated, as in the developing embryo (Mussini, Hutton,
and Udenfriend, 1967) and in the pregnant uterus
(Halme and Jaaskelainen, 1970), activity is increased.
High enzyme levels have also been found in other
collagen formative conditions such as healing
wounds and granulomas (Mussini et al, 1967), developing experimental cirrhosis (Takeuchi and Prockop,
1969), alcoholic liver disease in animals, (Feinman
and Lieber, 1972), and experimental pulmonary
fibrosis (Halme, Uitto, Kahanpaa, Karhunen, and
Linay, 1970). In acuteliver disease induced in mice it
has been shownthat thereis infact a roughparallelism
between hydroxylase activity and the rate of collagen
synthesis in liver (McGee, O'Hare, and Patrick, 1973).
On the basis of this evidence it is reasonable to assume that the high levels of collagen proline hydroxylase found in human cirrhosis in the present study
are probably a manifestation of a general increase in
fibroblastic biosynthetic activity. This interpretation of the results in the cirrhotic patients is in keeping with the finding that the hydroxylase level in
individual cirrhotics showed a good positive correlation with the degree of aggressive activity in each
case assessed morphologically; collagen formation
at the periphery of regenerating nodules is a prominent feature of progressive liver disease (Popper,
1966). The measurement of the hepatic level of this
enzyme may be, therefore, a useful quantitative index
of progression of the cirrhotic process in individual
patients. It may also prove to be helpful in assessing
the effect of therapeutic agents on collagen metabolism in chronic liver disease.
The increase in collagen proline hydroxylase
activity in the alcoholic patients and in the case of
persistent hepatitis without noteworthy fibrosis
requires discussion. If, as argued above, this observation is a reflection of a general increase in collagen
biosynthetic activity, it has to be postulated that
collagen degradative mechanisms at the time of
biopsy were capable of preventing a net morphological increase in collagen deposition. In experimental hepatic fibrosis collagen proline hydroxylase
activity begins to increase before fibrosis (McGee
and Patrick, 1972; McGee et al, 1973) or cirrhosis
(Takeuchi and Prockop, 1969) are morphologically
evident. If the latter experimental observations are
applicable to human liver disease the measurement
of hepatic collagen proline hydroxylase activity may
be of value in predicting which types of liver damage
may eventually progress to chronic liver disease and
cirrhosis. The group of patients under discussien is
being enlarged and followed up therefore to determine whether this is so.
Until recently it has been difficult to understand
why collagen proline hydroxylase should rise in
conditions characterized by increased collagen formation since it has not been shown that this enzyme
is rate limiting in collagen biosynthesis. The recent
finding that proline hydroxylation is required for the
assembly of a thermally stable triple helical collagen
molecule (Jimenez, Harsch, and Rosenbloom, 1973;
Berg and Prockop, 1973), however, indicates that
elevated hydroxylase levels in cirrhosis and other
collagen formative conditions is biologically necessary for the production of collagen molecules which
will not spontaneously denature at body temperature.
The results of the 35S sulphate investigation demonstrate that there is a very marked increase in
sulphated mucopolysaccharide synthesis in active
chronic liver diseases. Rather surprisingly it was
found that this increase was not confined to the areas
of connective tissue formation in the portal tracts
but was also prominent within hepatic sinusoids. It
would seem therefore that connective tissue biosynthesis is increased throughout the entire liver in
chronic progressive liver disease. Although autoradiography is a useful procedure for determining
the sites of intrahepatic fibrogenesis it suffers from
the disadvantage that it is not easily made quantitative and the results are not available for a period of
weeks. The results of collagen proline hydroxylase
measurements, however, are quantitative and are
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Collagen proline hydroxylase activity and 35S sulphate uptake in human liver biopsies
available about 2-5 hours after the biopsy has been
taken, while the tissue remaining is entirely adequate
for histopathological diagnosis.
We are grateful to Dr R. I. Russell and Professor
L. H. Blumgart of Glasgow Royal Infirmary for
their willing cooperation in providing the liver
biopsies used in this study. The work was supported
in part by a grant from the Medical Council on
Alcoholism.
References
Berg, R. A., and Prockop, I). J. (1973). The thermal transition of a
nonhydroxylated form of collagen. Evidence for a role for
hydroxyproline in stabilizing the triple-helix of collagen.
Biochem. biophys. Res. Commun., 52, 115-120.
Feinman, L., and Leiber, C. S. (1972). Hepatic collagen metabolism:
effect of alcohol consumption in rats and baboons. Science
(N. Y.), 176, 795.
Galambos, J. T. (1966). Acid mucopolysaccharides and cirrhosis of
the liver. Gastroenterology, 51, 65-74.
Grant, M. E., and Prockop, D. J. (1972). The biosynthesis of collagen.
New Engi. J. Med., 286, 194-300.
Halme, J., and Jaaskeliinen, M. (1970). Protocollagen proline hydroxylase of the mouse uterus during pregnancy and post-partum
involution. Biochem. J., 116, 367-369.
Halme, J., Uitto, J., KahanpAf, K., Karhunen, P., and Lindy, S.
(1970). Protocollagen proline hydroxylase activity in experimental pulmonary fibrosis of rats. J. Lab. clin. Med., 75, 535541.
Hutton J. J., Jr., Tappel, A. L., and Udenfriend, S. (1966). A rapid
assay for collagen proline hydroxylase. Analyt. Biochem., 16,
384-394.
Hutton, J. J., Jr., and Udenfriend, S. (1966). Soluble collagen proline
hydroxylase and its substrates in several animal tissues. Proc
nat. Acad. Sci. (Wash.), 56, 198-202.
Jimenez, S., Harsch, M., and Rosenbloom, J. (1973). Hydroxyproline
stabilizes the triple helix of chick tendon collagen. Biochem.
biophys. Res. Commun., 52, 106-115.
Lowry, 0. H., Rosebrough, N. J., Farr, A. L., and Randall, R. J.
267
(1951). Protein measurement with the Folin phenol reagent.
J. biol. Chem., 193, 265-275.
McGee, J. O.'D., Langness, U., and Udenfriend, S. (1971). Immunological evidence for an inactive precursor of collagen proline
hydroxylase in cultured fibroblasts. Proc. nat. Acad. Sci. (Wash.)
68, 1585-1589.
McGee, J. O'D., O'Hare, R. P., and Patrick, R. S. (1973). Stimulation
of the collagen biosynthetic pathway by factors isolated from
experimentally injured iliver. Nature [new Biol.], 243, 121-123.
McGee, J. O'D., ani Patrick, R. S. (1969a). The synthesis of sulphated mucopolysaccharide in mouse liver following carbon
tetrachloride injury. I. Autoradiographic studies. Brit. J. exp.
Path., 50, 521-526.
McGee, J. O'D., and Patrick, R. S. (1169b). The synthesis of sulphated mucopolysaccharide in (mouse liver following carbon
tetrachloride injury. II. Quantitation and partial characterization of extracted mucopolysaccharide. Brit. J. exp. Path., 50,
527-532.
McGee, J. O'D., and Patrick, R. S. (1972). The role of perisinusoidal
cells in hepatic fibrogenesis. Lab. Invest., 26, 429-440.
McGee, J. O'D., Rhoads, R. E., and Udenfriend, S. (1971). The substrate recognition site of collagen proline hydroxylase: the
hydroxylation of -x-Pro-Gly-sequences in bradykinin analogs
and other peptides. Arch. Biochem., 144, 343-351.
Miller, R. L., and Udenfriend, S. (1970). Hydroxylation of proline
residues in collagen nascent chains. Arch. Biochem., 139,
104-113.
Mussini, E., Hutton, J. J., Jr., and Udenfriend, S. (1967). Collagen
proline hydroxylase in wound healing, granuloma formation,
scurvy and growth. Science (N. Y.), 157, 927-929.
Popper, H. (1966). What is chronic hepatitis? Gastroenterology, 50,
4444 48.
Popper, H., and Udenfriend, S. (1970). Hepatic fibrosis: correlation
of biochemical and morphologic investigations. Amer. J. Med.,
49, 707-721.
Rhoads, R. E., and Udenfriend, S. (1970). Purification and properties
of collagen proline hydroxylase from newborn rat skin. Arch.
Biochem., 139, 329-339.
Takeuchi, T., and Prockop, D. J. (1969). Protocollagen proline hydroxylase in normal liver and in hepatic fibrosis. Gastroenterology, 56, 744-750.
Udenfriend, S. (1970). Biosynthesis of hydroxyproline in collagen. In
Chemistry and Molecular Biology of the Intercellular Matrix,
edited by E. A. Balazs, Vol. I, pp. 371-384. Academic Press,
London and New York.
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Collagen proline hydroxylase
activity and 35S sulphate uptake in
human liver biopsies
J. O'D. McGee, R. S. Patrick, Marion C. Rodger and
Carolyn M. Luty
Gut 1974 15: 260-267
doi: 10.1136/gut.15.4.260
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